Cell culture is widely used for the production of various harvestable bio-products including polypeptide growth factors, hormones, enzymes, vaccines and proteins. These bio-products are produced in culture by normal, transformed and genetically engineered cell lines.
When a cell is removed from its native environs, however, an artificial medium must provide all the critical factors necessary for normal cell growth, differentiation and host cell expression. Thus, the synthetic culture medium must supply critical nutritional, hormonal, and stromal factors normally provided in vivo. Furthermore, the culture medium should not interfere with the production or use of the desired bio-product. Since the function of many cell culture factors remain undefined at the molecular level, most cell lines used in the production of biologicals are grown in culture medium supplemented with animal serum which serves as a universal nutrient. Serum is a very complex fluid, containing at least 500 different protein components including hormones, growth factors, carrier proteins, trace elements and attachment and spreading factors among other constituents. Animal serum, such as fetal bovine serum (FBS), can represent up to 10% of a typical culture medium. Currently, C3A cells are grown in tissue culture medium containing approximately 5% FBS.
Although widely employed, serum supplementation has many limitations, particularly when utilized in the manufacture of biotechnology products (Barnes, 1987; Barnes & Sato 1980; Broad, i.e., 1991; Jayme, 1991). Serum contains high levels of numerous proteins that can interfere with heterologously expressed polypeptides and it must subsequently be separated from manufactured bio-products during their harvest. Additionally, the increasing rise in the price of commercial serum, the uncontrollable variation in its batch quality and the additional requirement for its sterilization, combine to complicate the production process and to inflate manufacturing costs.
Furthermore, serum use may present a potential bio-hazard. Serum can contain cryptic cytotoxic elements that must be removed before any biological product can be safely utilized after manufacture. Serious regulatory apprehension has been voiced by the United States Food and Drug Administration concerning the possible contamination of harvestable bio-products from cultures containing serum. Rossi, et al., (Am J. Vet. Res., 41:1680-1681 (1980)); Chu, et al., (In vitro, 9:31-34 (1973)); and Nuttall, et al., (Nature, 266:835-837 (1977)) address one aspect of this problem (viz., the potential for viral contamination of serum containing cell cultures). Viral contaminants may interact biologically with mammalian cells in culture, as noted by Chu, et al., or through obscure clinical reactions to other agents, as observed by Nuttall, et al.
A number of techniques have been employed to alleviate viral contamination of serum containing cultures. These include vigilant screening and eradication of contaminating viruses by affinity chromatography. Unfortunately, however, as reported by Orr et al., (J. Cain. Microbial., 3:402-5 (1976)), it is difficult to assure complete elimination of all possible undesirable agents that may exist in a contaminated cell culture. Recently, additional concerns have arisen concerning adulteration by agents of non-viral origin. Such fears have subsequently been confirmed by evidence of the transmission of prion-type diseases from animals to humans (e.g., neurodegenerative disorders such as Creutzfeldt-Jakob and most recently by a human form of Bovine Spongioform Encephalopathy; Collinge, J., Hum. Mol. Genet. 6: 1699-1705; 1997). These developments have raised further serious concerns over the use of animal-derived sera in cell culture.
Consequently, there is a high demand for the development of cell lines that are adapted for growth and culture in medium that is free of serum. Galfre, U.S. Pat. No. 4,350,683 describes a rat myeloma cell line (CNCM 1-078), useful in the development of rat-rat hybridomas, that is allegedly capable of growth in a serum-free medium. In many serum-free cultures, however, cell growth is often reported to be slower compared to cell growth in cultures supplemented with serum. Golde, U.S. Pat. No. 4,438,032 describes a human T-lymphoblast cell line (Mo) capable of growth in serum-free medium, although its rate of growth is considerably slower than when the cells are grown and maintained in medium containing 20% FBS. Furthermore, serum-free cell lines have also been reported to suffer from diminished cell density, decreased saturation levels, and reduced viability.
Consequently, there is a need to create serum-free cell lines that can grow and flourish when maintained in serum-free medium (including “chemically-defined” medium that is free of any type of serum or serum supplement). Such serum-free cell lines should also exhibit normal viability, rates of growth and be capable of expressing harvestable bio-products that are free of biological contamination.
Accordingly, the present invention offers a cell line—clonally derived from a parental C3A hepatocyte cell line—that is adapted for normal growth and maintenance in serum-free medium. Additionally, the present cell line supports the synthesis and expression, in serum-free culture, of substantial titers of harvestable bio-products at reasonably high purity including polypeptides and proteins. One advantage of the serum-free C3A cell line of the invention is that bio-products harvested from cultures utilizing these cells have considerably less risk of harboring cryptic infectious agents than bio-products manufactured with supplements containing serum. Another advantage is the likely facilitation of regulatory approval for bio-products derived using cells of the invention due to their reduced risk of contamination. One regulatory agency, the United States Department of Agriculture, is particularly concerned about the inadvertent introduction of Hoof and Mouth disease into United States livestock through importation of contaminated bio-products produced from cultures containing fetal calf serum. Consequently, the cell line of the invention and methods of its use offer advantages over cell lines that are grown or maintained in serum-containing medium.